Botulinum neurotoxin (BoNT) produced by neurotoxigenic clostridia are the most potent naturally occurring toxins known [Johnson, E. A. (2005) Topley and Wilson's Microbiology and Microbial Infections, eighth edition. 1035-1088]. Based on their antigenic specificity, BoNTs are distinguished into seven serotypes (A-G) [Gimenez, D. F., and Gimenez, J. A. (1995) Int. J. Food Microbiol. 27, 1-9], with BoNT/A, B and E accounting for nearly all recorded cases of human botulism [Johnson, E. A. (2005) Topley and Wilson's Microbiology and Microbial Infections, eighth edition, 1035-1088; Montecucco, C. and Molgo, J. (2005) Curr Opin Pharmacol. 5, 274-279]. BoNTs are zinc-containing metalloproteases of ca. 150 kDa consisting of a heavy chain (−100 kDa), and a light chain (˜50 kDa) linked by a disulfide bond.
The C-terminal domain of the heavy chain functions in receptor binding on the neuronal cell surface, inducing endocytotic internalization of the toxin. Once inside the endosome, protonation causes membrane insertion and chaperone/channel formation of the heavy chain coupled to light chain unfolding and entry into the channel. This is followed by light chain conduction through the heavy chain channel and subsequent release of the light chain by disulfide bond reduction and light chain refolding in the cytoplasm of the cell [4, 5]. Light chains of BoNTs are zinc endopeptidases that target core proteins including SNAP-25, VAMP/synaptobrevin, and syntaxin 1 involved in trafficking and release of neurotransmitters [Blasi, J., et al. (1993) Nature, 365:160-163; Binz, T., et al. (1994) J Biol. Chem. 269:1617-1620; Schiavo, G., et al. (1995) J. Biol. Chem. 268:11516-11519; Schiavo, G., et al., (1995) J. Biol. Chem. 270:10566-10570; Montecucco, C. and Schiavo, G. (1994) Mol. Microbiol. 13:1-8; Yamasaki, S., et al. (1994) J Biol. Chem. 269, 12764-12772].
The high potency of BoNT, its high specificity for motor neurons, and the longevity of its action (up to several months) have facilitated the use of BoNT/A and /B as extremely valuable drugs for treatment of a myriad of neurological diseases, as well as for cosmetic treatments, with BoNT/A being the most prominent serotype currently used [Foster, K. A., et al. (2006) Neurotox. Res. 9:133-140]. Despite the effective use of BoNTs in clinical applications, the major adverse effect has been the formation of antibodies which render patients refractory to treatment and tachyphylaxis [Borodic, G. (2007) Facial Plast. Surg. Clin. North Am. 15:11-16; Dressler, D. (2004) Mov Disord: 19(Suppl 8) S92-S100; and Borodic, G., et al. (1996) Neurology. 46:26-29]. For example, 5 to 10% of patients with cervical, segmental or multifocal dystonia receiving repeated BoNT/A treatments were estimated to develop resistance to treatments due to the presence of circulating neutralizing serum antibodies [Dressler, D. (2004) Mov Disord. 19(Suppl 8) S92-S100; Borodic, G., et al. (1996) Neurology. 46:26-29]. Resistance to BoNT treatment can be confirmed in a clinical setting by test injecting BoNT into the patient's frontalis muscle, extensor digitorum brevis (EDB) or sternomastoid muscle [Borodic, G. E. (1999) Current Opinions in Otolaryngology and Head and neck Surgery. 7:219-225; Borodic, G. E., et al. (1995) Neurology 45:204; Kessler, K. R. and Benecke, R. (1997) Mov Disord. 12:95-99; Cordivari, C., et al. (2006) Mov Disord., 21:1737-1741; Dressler, D. and Rothwell, J. C. (2000) Eur Neurol. 43:13-16], and measuring compound muscle action potentials. However, patients are not routinely monitored for antibody formation during their treatment regime, because a sensitive assay that measures neutralizing antibodies in human sera is not commercially available [Sesardic, D., et al. (2004) Mov Disord. 19 (Suppl 8): S85-91]. Such monitoring is highly desirable in clinical trials of BoNTs as well as for currently approved therapies.
Several laboratory assays for the detection of BoNTs and BoNT specific antibodies have been developed. The in vivo mouse bioassay currently is the standard method to detect BoNT activity, and the only assay approved by the FDA [Hatheway, C. L. (1988) Laboratory Diagnosis of Infectious Diseases. Principles and Practice. (Balows A., Hausler Jr. W. J., Ohashi M., Turano, A., Eds.) pp. 111-133. Springer-Verlag, New York; Schantz, E. J. and Kautter, D. A. (1978) J. Assoc. Off. Anal. Chem. 61:96-99]. In this assay, mice are injected intraperitoneally or intravenously with toxin or toxin/antibody mixtures and observed for signs of toxicity and death. While this assay is well-established and quantitative, it is relatively insensitive and has well-known drawbacks including the need for a large number of animals and associated required facilities and expenses, the requirement for 2-4 days for results, nonspecific deaths, and the need to expose mice to a high degree of pain and distress.
Alternative in vitro assays include the mouse diaphragm assay or MDA [Hatheway, C. L. (1988) Laboratory Diagnosis of Infectious Diseases. Principles and Practice. (Balows A., Hausler Jr. W. J., Ohashi M., Turano, A., Eds.) pp. 111-133. Springer-Verlag, New York], enzyme-linked immunosorbent assays (ELISAs) and variations, immunoprecipitation assay (IPA), chemiluminescent slot blot immunoassay, electro chemiluminscence, radioimmunoassay, lateral flow immunoassays, endopeptidase assays and others [Lindström, M. and Korkeala, H. (2006) Clinical Microbiology Reviews 19:298-314]. All of these assays can be used to quantitate BoNT's in vitro and in foods and clinical samples [Hatheway, C. L. (1988) Laboratory Diagnosis of Infectious Diseases. Principles and Practice. (Balows A., Hausler Jr. W. J., Ohashi M., Turano, A., Eds.) pp. 111-133. Springer-Verlag, New York; Sharma, S. K., et al. (2006) Appl Environ. Microbiol. 72:1231-1238; Sharma, S. K., et al. (2005) Appl. Environ. Microbiol. 71:3935-3941; and Sapsford, K. E., et al. (2005) Appl Environ Microbiol. 71:5590-5592]. However, many have the drawback of high background, and most measure only one biological property of BoNT activity (binding of the toxin to antibody, or proteolytic activity in the endopeptidase assays). In order to reliably measure BoNT holotoxin activity and detect neutralizing serum antibodies, an assay should simulate all aspects of intoxication (i.e.: binding of the heavy chain binding domain to the cell surface receptor, endocytosis, channel formation, conductance of the light chain into the cell's cytosoland disulfide bond cleavage, refolding of the light chain, and proteolytic cleavage of the target protein within the cell by the light chain).
A more complete approach for the screening of neutralizing antibodies as well as potency determination of the holotoxin is the use of cell-based BoNT assays. Several cell-based assays have been developed, including continuous cell lines such as neuro-2a, PC12, or SK-N-SH cells [Schiavo, G., et al. (1993) J. Biol. Chem. 268, 11516-11519; Dong, M., et al. (2004) PNAS 101:14701-14706; Yowler, B. C., et al. (2002) J. Biol. Chem. 277:32815-32819; Benatar, M. G., et al. (1997) J. Neuroimmunol. 80:1-5], as well as primary neurons derived from chicken, mouse or rat spinal cord cells [Stahl, A. M., et al. (2007) J. Biomol. Screen. 12:370-377; Hall, Y. H., et al. (2004) J. Immunol. Methods. 288:55-60; Keller, J. E., and Neale, E. A. (2001) J. Biol. Chem. 276:13476-13482; Keller, J. E., et al. (1999) FEBS Lett. 456:137-142; Keller, J. E., et al. (2004) Biochem. 43:526-532; Neale, E. A., et al. (1999) J. Cell. Biol. 147:1249-1260; Lalli, G., et al. (1999) J. Cell. Sci. 112:2715-2724; Welch, M. J., et al. (2000) Toxicon 38:245-258]. Successful detection of BoNT can be achieved by by Western blot assay of the cleaved target protein [Yowler, B. C., et al. (2002) J. Biol. Chem. 277:32815-32819; Keller, J. E., and Neale, E. A. (2001) J. Biol. Chem. 276:13476-13482; Keller, J. E., et al. (1999) FEBS Lett. 456:137-142; Keller, J. E., et al. (2004) Biochem. 43:526-532; Lalli, G., et al. (1999) J. Cell. Sci. 112:2715-2724], by specific FRET sensors [Dong, M., et al. (2004) PNAS 101:14701-14706], or by neuronal activity testing [Benatar, M. G., et al. (1997) J. Neuroimmunol. 80:1-5; Hall, Y. H., et al. (2004) J. Immunol. Methods. 288:55-60; Neale, E. A., et al. (1999) J. Cell. Biol. 147:1249-1260; Welch, M. J., et al. (2000) Toxicon 38:245-258]. However, continuous cell lines exhibit very low BoNT sensitivities and therefore cannot be used for detection of serum antibodies.
Most prior art primary neuronal cell assays using pure BoNT/A preparations have been reported to exhibit sensitivities of up to 50 μM of BoNT/A (˜250 to 750 mouse LD50 units), which is not sufficient for detection of most human serum antibodies. One primary cell assay has been adapted to detect as little as 3 μM BoNT/A and protection by up to 0.001 IU/ml of Equine International sera by measuring [3H] glycine release from primary rat spinal cord cells [Hall, Y. H., et al. (2004) J. Immunol. Methods. 288:55-60]. However, enhanced practicality and even higher sensitivity and specificity are desired for clinical and research applications.